The high degree of functional conservation of genes involved in the cell cycle combined with the superb cell biology and genetics of Drosophila make it an ideal model organism for studying cell cycle regulation. During early Drosophila embryogenesis, 3 genes are required for regulation of the streamlined "S-M" cycles characteristic of this developmental stage: pan gu (png), plutonium (plu), and giant nuclei (gnu). Females carrying mutations in any of these genes are sterile because their embryos fail to enter mitosis and over-replicate DNA to form "giant nuclei." PNG encodes a serine/threonine protein kinase, and plu and gnu encode small novel proteins, png, plu, and gnu interact genetically with cyclin B, and Cyclin B protein levels are decreased in these mutants. We recently demonstrated that PNG, PLU, and GNU interact to form an active kinase complex in vitro and that GNU activates PNG by inducing dimerization of PNG-PLU complexes. Additionally, we showed that GNU itself is a substrate for PNG kinase. In a genome-wide biochemical screen, several PNG kinase substrates with vertebrate homologs were recently identified, suggesting that the PNG kinase complex regulates the cell cycle by impinging on evolutionarily conserved components. We now propose to determine whether GNU activates PNG kinase via a dimerization mechanism in vivo and to test whether it is a substrate with potential function as an effector. We plan to determine whether the PNG kinase complex regulates activity of the Anaphase Promoting Complex so as to control Cyclin B levels and entry into mitosis. Finally, an evolutionarily conserved protein, Mat89Bb, has been identified as an in vitro PNG kinase substrate. RNA interference of Mat89Bb in Drosophila results in a phenotype reminiscent of that of the giant nuclei class of genes. We will test whether Mat89Bb is an in vitro substrate of PNG and will isolate Mat89Bb mutants to more precisely determine its role in cell cycle regulation.